human crispr pooled genome-wide brunello sgrna library  (Addgene inc)

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) Inhibition of anticancer immunity by Siglec receptors. Siglecs bind to sialic acid-containing glycans on the surface of target cells. B) Components of the Siglec-7 ligand structure. Disialyl-T glycans are organized in dense arrays on the backbone of specific mucin-type O-glycoproteins. C ) Targeted overexpression of genes by CRISPRa. A cell line expressing a dCas9-GCN4 peptide array and a VP64-SunTag fusion protein serves as the target cell line (K-562-CRISPRa cells). This cell line is lentivirally transduced with an sgRNA targeting the promoter region of a target gene. Subsequent recruitment of chromatin remodeling factors induces an increase in transcriptional activity. D) Recombinant Siglec-Fc proteins were precomplexed with an AlexaFluor647-antihuFc antibody (1 μg/mL) for 1 hour on ice. Precomplexes were subsequently incubated with K-562 cells for 30 minutes and analyzed by flow cytometry. Representative flow cytometry plots are depicted for each Siglec-Fc as well as a human Fc (hFc) negative control. E) K-562-CRISPRa cells were lentivirally transduced with a genome-wide library of ∼104,000 sgRNAs (5 sgRNAs/gene). After selection and propagation, 1.25 x 10 8 cells were then stained with Siglec-Fc reagents as in B . A high-binding (top 20%) and low-binding (bottom 20%) of the fluorescent population was then selected and sorted by FACS.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Inhibition, Over Expression, Expressing, Peptide Microarray, Transduction, Activity Assay, Recombinant, Incubation, Flow Cytometry, Negative Control, Genome Wide, Selection, Staining, Binding Assay

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) K-562-dCas9-CRISPRa cells were transduced with two different sgRNAs targeting the CD24 promoter region. After selection with puromycin, cells were stained with Siglec-10-Fc as in 1B and with a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. B) THP-1 cells were stained with Siglec-10-Fc as in 1B and a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. C) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting MGAT1 to generate a polyclonal cell population containing WT and MGAT1 KO cells. Cells were then co-stained with Siglec-10-Fc and the lectin L-PHA (5 μg/mL), which binds to complex N-linked glycans. Representative staining is indicated on the flow cytometry plot. D) Graph indicates MFI of Siglec-10-Fc staining in the WT (L-PHA+) and MGAT1 KO (L-PHA-) populations. E) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting C1GALT1. Cells were co-stained with Siglec-10-Fc and the lectin DBA, which binds to exposed α-GalNAc. Representative staining is indicated on the flow cytometry plot. F) Graph indicates MFI of Siglec-10-Fc staining in the WT (DBA-) and C1GALT1 KO (DBA+) populations. G) Pathway diagram indicates the enzymes involved in 2,3-linked and 2,6-linked sialylation of N-linked glycans. ST3GAL4, ST3GAL6 and ST6GAL1 were all strong hits in the Siglec-10 screen. H) The heat map indicates the average mRNA expression of key glycosyltransferase hits in cell lines derived from B-ALL, T-ALL, AML and multiple myeloma. mRNA expression values were extracted from DepMap and the Human Protein Atlas. I) OCI-AML-2-Cas9 cells were transduced an sgRNAs against ST3GAL4. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. J) The MFI of Siglec-10 staining in OCI-AML-2 WT and ST3GAL4 KO cells is indicated. MFI is internally normalized to the WT cell line. K) MM1S-Cas9 cells were transduced an sgRNAs against ST6GAL1. ST6GAL1 KO cells were isolated by FACS using the lectin SNA. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. L) The MFI of Siglec-10 staining in MM1S WT and ST6GAL1 KO cells is indicated. MFI is internally normalized to the WT cell line. M) K-562-CRISPRa cells were transduced and stained as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CD44 High” expression gate. N) The MFI of Siglec-10-Fc staining in NT cells vs. “CD44 High” cells is shown. MFI is internally normalized to the WT cell line. O) K-562 CRISPRa cells were transduced with an sgRNA against CSPG4 and co-stained with Sig10-Fc and a CSPG4 antibody as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CSPG4 High” expression gate. P) The MFI of Siglec-10 staining in NT cells vs. “CSPG4 High” cells is shown. MFI is internally normalized to the WT cell line. Statistical significance was determined using a two-tailed t-test. ** indicates p<0.01, * indicates p<0.05. Mean values plotted, error bars indicate SEM.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Transduction, Selection, Staining, Flow Cytometry, Expressing, Derivative Assay, Labeling, Isolation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: RBM39 shapes innate immunity by controlling the expression of key factors of the interferon response

doi: 10.3389/fimmu.2025.1568056

Figure Lengend Snippet: A genome-wide CRISPR/Cas9 screen identifies potential TLR3-related factors. (A) Schematic of the lentiviral truncated BID (tBID) death reporter system. The tBID expression is driven by the IFIT1 promoter, while the expression of the neomycin selection marker (NeoR) is under the control of the constitutive PGK promoter. Working principle: (1) dsRNA is taken up via endocytosis and transported to the endosome. (2) The endosomal TLR3 recognizes dsRNA and triggers signaling via TRIF, resulting in the activation of transcription factors IRF3 and NF-κB. (3) Activated transcription factors induce the expression of tBID by binding the IFIT1 promoter. (4) tBID associates with Bcl-2 proteins BAX and BAK to form a complex that permeabilizes the outer mitochondrial membrane and mediates the release of cytochrome c (Cyt c) into the cytoplasm. (5) Cyt c binds APAF1, triggering its oligomerization and binding to Pro-Caspase-9. Activated Caspase-9, in turn, activates Caspase-3 and -7, inducing apoptosis. (B) Workflow of the CRISPR/Cas9 screen. PH5CH and Huh7-Lunet-TLR3 stably expressing the death reporter and Cas9 were transduced with the genome-wide lentiviral CRISPR sgRNA library and selected with puromycin for stable expression. TLR3 stimulation with poly(I:C) induced the expression of tBID and thus apoptosis in case of intact TLR3 signaling. Then, surviving TLR3-deficient cells were enriched and collected for next-generation sequencing (NGS). (C) Illustration of the filtering steps to generate the final hit list. For each experiment independently, the ~2,000 most enriched gRNAs were determined in the NGS dataset and matched to their respective target genes, creating a list of 2,154 initial genes of interest. Subsequently, it was only focused on protein-coding genes. To ensure reproducibility, only genes with enriched gRNAs in three out of four repetitions were considered; this also eliminated genes showing enrichment in only one of the cell lines used. Lastly, genes were filtered to have at least five out of six available gRNAs enriched in at least one repetition to remove hits driven by potential off-target effects of just a subset of gRNAs. (D–F) A total of 50 candidate genes were selected and subjected to siRNA silencing for 48 h; then, the cells were stimulated with 50 μg/mL poly(I:C) supernatant feeding for 8 h. IFIT1 mRNA was measured by RT-qPCR normalized to GAPDH and expressed as relative expression to siRNA non-targeting (siNT)-treated samples. Knockdown of the candidates upregulated (D) or downregulated (E) IFIT1 mRNA expression in Huh7-Lunet-TLR3 cells. Knockdown of RBM39 , PCF11 , PTPRT , and KDM2A in PH5CH cells reduced IFIT1 mRNA expression in PH5CH cells (F) . siUNC93B1, siTLR3, and siTRIF were used as positive controls. The data are from three biological replicates ( n = 3); error bars indicate standard deviation (SD).

Article Snippet: After that, PH5CH and Huh7-Lunet-TLR3 cells stably expressing the tBID death reporter and Cas9 were transduced with lentiviral vectors containing the GeCKOv2.0 human-genome-wide sgRNA library ( ) (Addgene, USA) at MOI = 0.3.

Techniques: Genome Wide, CRISPR, Expressing, Selection, Marker, Control, Activation Assay, Binding Assay, Membrane, Stable Transfection, Transduction, Next-Generation Sequencing, Quantitative RT-PCR, Knockdown, Standard Deviation

Journal: Scientific reports

Article Title: CRISPR-Cas9 genetic screens reveal regulation of TMPRSS2 by the Elongin BC-VHL complex.

doi: 10.1038/s41598-025-95644-0

Figure Lengend Snippet: Fig. 3. Genome-wide and targeted CRISPR-Cas9 screens reveal Elongin B and VHL as regulators of TMPRSS2 expression. (A) Schematic workflow of the genome-wide and targeted CRISPR-Cas9 screens. Caco-2 cells were transduced with lentivirus encoding CRISPR library followed by selection with puromycin. The populations of cells with low TMPRSS2 expression were enriched by FACS with the TMPRSS2-specific antibody, subjected to genomic DNA isolation followed by Illumina sequencing of the integrated gRNAs. (B, C) MAGecK RRA (robust rank aggregation) scores of the enriched sgRNAs from the CRISPR-Cas9 screens performed with the genome-wide sgRNA library (B) and sgRNA library targeting Epigenetic Modifiers and Transcriptional Regulators (C).

Article Snippet: Genetic screens with genome-wide and Epigenetic modifiers and transcriptional regulators CRISPR sgRNA libraries Human Improved Genome-wide Knockout CRISPR Library v1 was a gift from Kosuke Yusa (Addgene #67989)73.

Techniques: Genome Wide, CRISPR, Expressing, Transduction, Selection, DNA Extraction, Illumina Sequencing